It is fairly well established that many pathological conditions, such as infections, cancer, autoimmune disorders, etc., are characterized by the inappropriate expression of certain molecules. These molecules thus serve as "markers" for a particular pathological or abnormal condition. Apart from their use as diagnostic "targets," i.e., materials to be identified to diagnose these abnormal conditions, the molecules serve as reagents which can be used to generate diagnostic and/or therapeutic agents. A by no means limiting example of this is the use of cancer markers to produce antibodies specific to a particular marker. Yet another non-limiting example is the use of a peptide which complexes with an MHC molecule, to generate cytolytic T cells against abnormal cells.
Preparation of such materials, of course, presupposes a source of the reagents used to generate these. Purification from cells is one laborious, far from sure method of doing so. Another preferred method is the isolation of nucleic acid molecules which encode a particular marker, followed by the use of the isolated encoding molecule to express the desired molecule.
To date, two strategies have been employed for the detection of such antigens in, e.g., human tumors. These will be referred to as the genetic approach and the biochemical approach. The genetic approach is exemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA, 85:2275 (1988), incorporated by reference. In this approach, several hundred pools of plasmids of a cDNA library obtained from a tumor are transfected into recipient cells, such as COS cells, or into antigen-negative variants of tumor cell lines. Transfectants are screened for the expression of tumor antigens via their ability to provoke reactions by anti-tumor cytolytic T cell clones. The biochemical approach, exemplified by, e.g., Mandelboim et al., Nature, 369:69 (1994) incorporated by reference, is based on acidic elution of peptides which have bound to MHC-class I molecules of tumor cells, followed by reversed-phase high performance liquid chromography (HPLC). Antigenic peptides are identified after they bind to empty MHC-class I molecules of mutant cell lines, defective in antigen processing, and induce specific reactions with cytotoxic T-lymphocytes. These reactions include induction of CTL proliferation, TNF release, and lysis of target cells, measurable in an MTT assay, or a .sup.51 Cr release assay.
These two approaches to the molecular definition of antigens have the following disadvantages: first, they are enormously cumbersome, time-consuming and expensive; second, they depend on the establishment of cytotoxic T cell lines (CTLs) with predefined specificity; and third, their relevance in vivo for the course of the pathology of disease in question has not been proven, as the respective CTLs can be obtained not only from patients with the respective disease, but also from healthy individuals, depending on their T cell repertoire.
The problems inherent to the two known approaches for the identification and molecular definition of antigens is best demonstrated by the fact that both methods have, so far, succeeded in defining only very few new antigens in human tumors. See, e.g., van der Bruggen et al., Science, 254:1643-1647 (1991); Brichard et al., J. Exp. Med., 178:489-495 (1993); Coulie et al., J. Exp. Med., 180:35-42 (1994); Kawakami et al., Proc. Natl. Acad. Sci. USA, 91:3515-3519 (1994).
Further, the methodologies described rely on the availability of established, permanent cell lines of the cancer type under consideration. It is very difficult to establish cell lines from certain cancer types, as is shown by, e.g., Oettgen et al., Immunol. Allerg. Clin. North. Am., 10:607-637 (1990). It is also known that some epithelial cell type cancers are poorly susceptible to CTLs in vitro, precluding routine analysis. These problems have stimulated the art to develop additional methodologies for identifying cancer associated antigens.
One key methodology is described by Sahin et al., Proc. Natl. Acad. Sci. USA, 92:11810-11913 (1995), incorporated by reference. Also, see U.S. patent application Ser. No. 08/580,980, filed on Jan. 3, 1996 and U.S. Pat. No. 5,698,396. All three of these references are incorporated by reference. To summarize, the method involves the expression of cDNA libraries in a prokaryotic host. (The libraries are secured from a tumor sample). The expressed libraries are then immunoscreened with absorbed and diluted sera, in order to detect those antigens which elicit high titer humoral responses. This methodology is known as the SEREX method ("Serological identification of antigens by Recombinant Expression Cloning"). The methodology has been employed to confirm expression of previously identified tumor associated antigens, as well as to detect new ones. See the above referenced patent applications and Sahin et al., supra, as well as Crew et al., EMBO J., 144:2333-2340 (1995).
The SEREX methodology has been applied to esophageal cancer samples, and an esophageal cancer associated antigen has now been identified, and its encoding nucleic acid molecule isolated and clones, as per U.S. patent application Ser. No. 08/725,182 filed Oct. 3, 1996, incorporated by reference herein.
The relationship between some of the tumor associated genes and a triad of genes, known as the SSX genes, was investigated by Sahin et al., supra, and Tureci et al., Cancer Res., 56:4766-4772 (1996). For example, one of these SSX genes, referred to as SSX-2, was identified, at first, as one of two genes involved in a chromosomal translocation event (t(X; 18) (p11.2; q 11.2)), which is present in 70% of synovial sarcomas. See Clark et al., Nature Genetics, 7:502-508 (1994); Crew et al., EMBO J., 14:2333-2340 (1995). It was later found to be expressed in a number of tumor cells, and is now considered to be a tumor associated antigen referred to as HOM-MEL-40 by Tureci et al., supra. Its expression to date has been observed in cancer cells and normal testis only. Thus, parallels other members of the "CT" family of tumor antigens, since they are expressed only in cancer and testis cells. Crew et al. also isolated and cloned the SSX-1 gene, which has 89% nucleotide sequence homology with SSX-2. See Crew et al., supra. Additional work directed to the identification of SSX genes has resulted in the identification of SSX-3, as is described by DeLeeuw et al., Cytogenet. Genet., 73:179-183 (1996). The fact that SSX presentation parallels other, CT antigens suggested to the inventors that other SSX genes might be isolated. See Gure et al., Int. J. Cancer, 72:965-971 (1997), incorporated by reference.
Application of a modification of the SEREX technology described, supra, has been used, together with other techniques, to clone two, additional SSX genes, referred to as SSX4 and SSX5 hereafter, as well as, an alternate splice variant of the SSX4 gene. Specifically, while the SEREX methodology utilizes autologous serum, the methods set forth infra, use allogenic serum. See U.S. patent application Ser. No. 08/851,138, filed May 5, 1997, incorporated by reference.
These investigations have all led to the identification of antigens associated with cancer and, in many cases, the isolation of previously unknown molecules. Exemplary of these are MAGE-1, which is disclosed in, e.g., U.S. Pat. No. 5,342,774 and van der Bruggen et al., Science, 254:1643-1647 (1991), incorporated by reference. These references also disclose MAGE-3, which is also described in allowed U.S. patent application Ser. No. 08/037,230, filed Mar. 26, 1993, incorporated by reference.
With respect to the members of the SSX family discussed herein, SSX-1 is disclosed by Crew et al., EMBO J., 14:2333-2340 (1995), incorporated by reference. The SSX-2 gene is disclosed by Clark et al., Nature Genetics, 7:502-508 (1994). Independently, Pfreundschuh identified the so-called "HOM-MEL 40" gene as a cancer associated antigen, as is described in U.S. Pat. No. 5,698,396, also incorporated by reference. The SSX4 gene is described in Ser. No. 08/851,138, filed Mar. 5, 1997 and incorporated by reference. Also see Gure et al., Int. J. Cancer, 72:965-971 (1997), incorporated by reference. With respect to SCP-1, see U.S. patent application Ser. No. 08/892,702 filed Jul. 15, 1997, and incorporated by reference, and Meuwissen et al., EMBO J., 11(13):5091-5100 (1992), also incorporated by reference. For NY-ESO-1, see allowed U.S. patent application Ser. No. 08/725,182, filed Oct. 3, 1996, and Chen et al., Proc. Natl. Acad. Sci. USA, 94:1914-1918 (1997), incorporated by reference. BAGE, another of the antigens in this family, is described in U.S. Pat. No. 5,571,711, and Boel et al., Immunity, 2:167-175 (1995), incorporated by reference. The GAGE antigen is described, e.g., in U.S. Pat. Nos. 5,610,013 and 5,648,226, both of which are incorporated by reference. Several members of the GAGE family are described in these references; however, due to their high degree of homology, GAGE is used in the singular hereafter to refer to this family of related molecules because in the experiments which follow, the primers used will identify all forms of the gene.
Expression of these antigens is associated with cancer; however, there is no antigen, including the specific antigens discussed herein, which have been found to be expressed in all tumors. Further, there has not been any tumor type observed, wherein expression of the antigen is ubiquitous. Hence, further efforts are needed to correlate expression of these antigens with tumor types.
It has now been found that by carrying out multiple assays, one can develop a clearer idea of whether or not breast cancer or melanoma is present in a patient sample. This diagnostic approach is one feature of the invention. This, in turn, has therapeutic implications, because one can develop therapeutic approaches such as peptide cocktails, based upon the pattern of expression found in a particular tumor sample. This can include, e.g., the mixing of tumor rejection antigens, which are peptides derived from the longer antigens and are known to associate with MHC molecules, provoking cytolytic T cell line proliferation use of such peptides requires HLA typing of the subject under consideration, but this is a standard technique well-known to the art. Further, one can compare the expressed antigens to determine regions of homology within tumor rejection antigen sequences, so as to determine the smallest number of so-called "TRAs" necessary to provoke a response.
These, and other aspects of the invention, will become clear from the disclosure which follows: